F-Tracker3D: tracking fluorescent cells in three dimensions

OBJECTIVE: Nowadays, the usage of multicellular in vitro models and microscopy techniques optimized for 3D analyses is blooming in the biological laboratories. It has been extensively proved that 3D models represent a more reliable testbed than 2D ones for studying new drugs, cell-cell interaction, and cell-biomaterial compatibility. However, today just few software tools are available for analysing 3D images and none of them is a user-friendly tool for tracking cells. This prevents, for instance, the possibility to monitor the motility of cells seeded on a biomaterial scaffold. In order to provide biologists with a turnkey solution to perform analyses of cell motility in 3D, we designed Fluorescent Cell Tracker in 3D (F-Tracker3D), a user-friendly software tool for tracking individual fluorescent-tagged proteins and cells directly in 3D. MATERIALS AND METHODS: F-Tracker3D is an open-source software tool available at: https://sourceforge.net/p/ftracker3d. It provides automatic, semi-automatic and manual methods to track single particles/cells in time-lapse confocal/light-sheet fluorescent microscopy (LSFM) acquisitions. No image processing skills or prior knowledge of the sample is needed. For each tracked cell, F-Tracker3D automatically computes (x, y, z) for each time point t. It provides several measurements computed according to the spatial cell displacement, and an early version of the software was recently used to quantitatively monitor the cell displacement of Mesenchymal Stromal Cells (MSCs) loaded onto silk-fibroin coated alginate beads, so to evaluate the material’s biocompatibility. RESULTS: By combining LSFM and F-Tracker3D we were able to demonstrate that the motility of MSCs onto fibroin-coated alginate scaffolds one day and three days after seeding significantly differs. One day after seeding, the MSC’s shape is rounded, typically referring to cells not yet completely adherent to the surface material, and the cell motion is basically random. After 3 days, MSCs fully attach to the fibroin coating, showing a polarized orientation for motility with the aim of covering the entire surface of the beads. These data suggest that the analysis of cell-biomaterial interactions should be performed several days after cells seeding to permit a perfect cell adhesion and elongation along the surface of the desired biomaterial. CONCLUSION: In this work, we described F-Tracker3D, an open-source user-friendly software tool we developed for monitoring cells on 3D scaffolds. By using F-Tracker3D we were able to quantify the MSC’s adhesion capacity on a silk-fibroin coated alginate material and detect differences in cell motility along the scaffold surface. Moreover, we were also able to produce a holistic view of the cell distribution during time. We therefore propose the combination of LSFM and F-Tracker3D as a tool to design tissue engineering products, as well as to perform quality control during validation of scaffolds to be used as medical devices.